S285: DISSECTING THE ROLE OF THE IMMUNOPHILIN FKBP12 IN HEPCIDIN REGULATION
Authors: Mariateresa Pettinato, Alessandro Dulja, Silvia Colucci, Valeria Furiosi, Franca Fette, Andrea Steinbicker, Martina Muckenthaler, Antonella Nai, Alessia Pagani, Laura Silvestri
Journal: HemaSphere (2023)

Background: The liver hormone hepcidin is the main regulator of iron metabolism. It controls body iron levels by blocking the cellular iron exporter ferroportin in response to increased serum and liver iron, and its deficiency is a common hallmark of hemochromatosis and diseases caused by ineffective erythropoiesis. Hepcidin expression is modulated by the BMP-SMAD pathway in response to BMP2 and the iron-responsive ligand BMP6. Upon ligand binding, the type-II receptors ACVR2A and BMPR2 activate the type-I receptors ALK2 and ALK3, leading to phosphorylation and nuclear translocation of SMAD1/5/8 and thus hepcidin expression. Iron deficiency and/or increased erythropoiesis reduce hepcidin expression mainly through the serine protease TMPRSS6 and erythroferrone. We recently identified the immunophilin FKBP12 as a new hepcidin inhibitor. FKBP12 acts by blocking ALK2 and can be displaced from the receptor by both the physiologic ALK2 ligand BMP6 and the immunosuppressive drug tacrolimus (TAC) (Colucci et al., Blood 2017). Aims: The aim of the study is to dissect the molecular mechanism(s) whereby FKBP12 regulates the BMP-SMAD pathway and hepcidin expression Methods: Human hepatoma cell line HuH7 was used to investigate with which receptors FKBP12 interacts and how FKBP12 regulates the interaction between type-I and type-II receptors by co-immunoprecipitation experiments. In primary murine hepatocytes Fkbp12, BMPRI and BMPRII were silenced in primary murine hepatocytes isolated from wild type mice to investigate the effect of TAC and BMP6 on hepcidin/BMP-SMAD signaling. Finally, we tested the synergy between TAC and BMP6 was analyzed both in vitro in HuH7 cells and in vivo in Hjv KO mice chronically treated for 28 days with suboptimal TAC administered through miniosmotic pump delivery of the drug. Results: FKBP12 specifically interacts with ALK2, but not with ALK3 or BMPRII, in hepatoma cells. Interestingly, mutations in ALK3 that reduce or block its activation can rescue the binding of FKBP12 with ALK3. This suggests that the catalytic activation of ALK3 in hepatocytes inhibits its interaction with FKBP12 and explains why FKBP12 exhibits a cell-specific affinity for type I receptors. In murine primary hepatocytes, Fkbp12 silencing increases hepcidin and Id1 expression and abrogates the TAC-dependent hepcidin upregulation, demonstrating that TAC modulates the BMP pathway and hepcidin exclusively via FKBP12. Additionally, the activation of the BMP-SMAD pathway by TAC is prevented by silencing of Alk2 and Acvr2a, but not Bmpr2. Alk3 silencing also strongly reduces the TAC effect. Given that BMP6 signals preferentially through ALK2 and reduces ALK2-FKBP12 interaction, we hypothesized that BMP6 would act through the same receptors. In agreement, the response to BMP6 is blunted in Alk2 and Acvr2a silenced hepatocytes, and strongly impaired in Alk3 deficient cells. Mechanistically, both TAC and BMP6, by displacing FKBP12 from ALK2, increase ALK2 homo- and ALK2-ALK3 hetero-oligomerization, and favor the interaction of ALK2 with type-II receptors. By acting on the same receptors, TAC and BMP6 cooperate in activating the BMP pathway and hepcidin expression both in vitro and in vivo in Hjv KO mice. Summary/Conclusion: We demonstrate that FKBP12 inhibits the BMP-SMAD pahtway by binding ALK2 and preventing its functional interaction with ALK3, ACVR2A and BMPR2. Decreasing the binding of FKBP12 to ALK2 could be a novel approach worth to be explored to increase, at least partially, hepcidin expression in hemochromatosis or in disorders with ineffective erythropoiesis, all conditions characterized by high BMP6 levels. Keywords: Iron metabolism, Hepcidin